RSV infection has been a longstanding and pernicious problem globally, including the United States, Europe, Australia and Japan. It is particularly troublesome in premature infants, young children, and the elderly, and indeed for all individuals with a weakened immune system. It is estimated that about two thirds of children below age 1 and almost all children between age 1 and 4 are infected at least once with RSV, with most recovering without any need for medical attention. However, 5-10% have prolonged severe infection, a factor believed to be predisposing to wheezing and asthma-like symptoms later in childhood. RSV has two major surface glycoproteins, F and G. The sole marketed monoclonal antibody against RSV is only approved for prophylactic use in premature infants to prevent infection by RSV, and is directed against the F protein. This antibody, palivizumab (Synagis®, from Medlmmune) is broadly useful due to conservation of the F protein sequence among strains. By contrast, the G protein is quite variable except for a central “CX3C” domain that is nearly invariant in nearly 100 sequenced strains. This region includes a motif that has been shown to interact with the fractalkine receptor. That interaction is believed to contribute to the prolonged disease course characteristic of RSV by suppressing an effective immune response to the virus: Tripp, R. A, et al., Nature Immunology (2001) 2:732-738. This region has also been shown to be an antagonist of the Toll-like Receptor 4, which is again believed to contribute to suppressing an effective immune response: Polack, et al., Proc. Natl. Acad. Sci. USA (2005) 102:8996-9001; Shingai, et al., Int'l Immunology (2008) epub July 8.
Initial attempts at prophylaxis for RSV by vaccination proved counterproductive. Enhanced disease and pulmonary eosinophilia were associated with vaccination with formalin inactivated RSV or with RSV G glycoprotein and this has been attributed to the above noted conserved sequence in the G protein designated CX3C region which mimics the chemokine fractalkine. (Haynes, L. M., et al., J. Virol. (2003) 77:9831-9844.) Passive immunization using antibodies directed to the G protein has generally been considered impractical due to the lack of conservation of the sequence of this protein among strains.
It has subsequently been confirmed by the same group that anti-G protein antibody responses engendered by RSV infection or vaccination are associated with inhibition of the binding of the G protein to the fractalkine CX3C receptor and with modulation of RSV G-protein-mediated leukocyte chemotaxis (Harcourt, J. L., et al., J. I. D. (2004) 190:1936-1940) and that inhibition of this binding adversely affects T cell responses (Harcourt, J. L., et al., J. Immunol. (2006) 176:1600-1608). More recent vaccine efforts have avoided the worsening of disease associated with the formalin fixed vaccine, but the immunity conferred by the newer vaccines has been found to wane rapidly (weeks to months), consistent with the poor immunological memory to natural RSV: Yu, et al., J. Virol. (2008) 82:2350-2357. Repeated infection is common for this virus, unlike many others. The immunosuppressive properties of the G protein may be responsible for this effect.
Monoclonal antibodies directed against the G protein have been known for over 20 years. Anderson, L. J., et al., J. Virol. (1988) 62:4232-4238 describe the ability of mixtures of F and G protein monoclonal antibodies (mAbs), and of the individual mAbs, to neutralize RSV. The mAbs relevant to binding G protein, notably 131-2G, were later studied by Sullender, W., Virol. (1995) 209:70-79 in an antigenic analysis. This antibody was found to bind both RSV groups A and B, representing the major strains of RSV.
In addition, Mekseepralard, C., et al., J. Gen. Virol. (2006) 87:1267-1273 summarize earlier papers showing that passively administered antibodies both to F and G protein were protective against experimental infection in rodent models. These articles include Routledge, et al., J. Gen. Virol. (1988) 69:293-303; Stott, E. J., et al., J. Virol. (1986) 60:607-613; Taylor, G., et al., Immunol. (1994) 52:137-142; and Walsh, E. E., et al., Infect. Immun. (1984) 43:756-758. In the instant article, Mekseepralard, et al., noted that a specific monoclonal antibody raised against the G protein (1C2) required glycosylation in order to neutralize the virus in the presence of complement in vitro or when used in vivo in mice. The authors note that amino acids 173-186 of the G protein are conserved and that 1C2 was directed against a conserved region; however, the method for preparing non-immunogenic antibodies was relatively crude, namely chimerization of a murine Fab onto a human Fc region.
In addition, Corbeil, S., et al., Vaccine (1996) 14:521-525 demonstrate that the complement system is involved in the protection of mice from challenge with RSV after passive immunization with the murine monoclonal antibody 18A2B2, even though this antibody does not show neutralizing capability in vitro.
PCT publication WO 00/43040 describes the use of anti-Substance P antibodies in ameliorating the airway inflammation associated with infection by RSV. The production of Substance P, a known proinflammatory mediator, is enhanced by administration of the G protein of RSV and is absent in mutants of RSV that are missing the G protein or carry a function defeating point mutation in the central conserved region: Haynes, et al., J Virol (2003) 77:9831-9844.
U.S. patent publication 2006/0018925 describes and claims antibodies and small peptides that are able to block the interaction of CX3C region of the G protein with its receptor. These compositions are suggested as useful for modulating RSV infection and inducing immunity. Although humanization of the murine antibodies employed in the demonstration of the therapeutic and prophylactic value of these antibodies is suggested, no such humanized forms were actually produced or described.
PCT publication WO2007/101441, assigned to Symphogen, is directed to recombinant polyclonal antibodies for treatment of RSV infections. The polyclonal recombinant antibodies are composed of individual monoclonal antibodies that were isolated from human serum. Table 5 of this publication describes 12 monoclonal antibodies that are said to bind to a “conserved region” at amino acids 164-176 of the RSV G protein of subtype A. Five of these were tested for affinity to the G protein and affinities in the range of 100-500 pM were found. Two of these antibodies were tested for neutralizing ability using the plaque reduction neutralization test (PRNT); one showed an EC50 value of approximately 2.5 μg/ml and the other failed to display neutralization characteristics at all.